We shall be studying the mechanism whereby cultured animal cells become resistant to methotrexate as a result of selective amplification of DNA sequences coding for dihydrofolate reductase. The research is directed at the fundamental mechanism(s) whereby this occurs and results in amplified DNA sequences that are either present on chromosomes, or are present at extrachomosomal elements called "double minute chromosomes." Gene amplification occurs on evolutionary time scale, as well as one mechanism for developmental regulatino of protein synthesis. We wish to use the experimental system of cultured mammalian cells to study the mechanism of amplification. The studies will employ varoius cells lines of cultured hamseter and mouse cells. Mechanisms to be studied include uptake-replicaton-integration of DNA from cells, as well as a saltatory replication model. Techniques to be employed include use of fluorescence-activated cell sorter to study DNA synthesis, as well as isolate cells with elevated dihydrofolate reductase levels. Dihydrofolate reductase DNA sequences will be detected and isolate employing both messenger-derived, as well as genomic recombinant DNA clones presently available in the laboratory. Ultimately, we are interested in studing how genes are amplified, and what intrinsic and extrinsic factors may affect the amplification process.